Reaction of Radicals with Tirapazamine
A R T I C L E S
which explains the reshaping of the band in this region observed
in Figure 6. Our assignment of the transient absorption growing
at 350 and 400 nm is also consistent with the previous pulse
radiolysis-transient spectroscopy work of Anderson and co-
workers.5
The putative H atom transfer reaction of ketyl radical from
the OH moiety is significantly more exothermic (9.7 kcal/mol)
than the corresponding reaction of tert-butyl and dioxanyl
radicals from a C-H bond adjacent to the radical center. The
same idea explains the large difference in reactivity between
the radicals derived from triethylamine and diisopropylamine.
In the latter case, the radical can undergo a hydrogen atom
transfer, similar to that possible with a ketyl radical, and has 3
times as many â-hydrogen atoms as the triethylamine-derived
radical which are able to react with TPZ.
butyl peroxide (DTBP) in the same solvents. The ketyl radicals
react rapidly with tirapazamine (TPZ). The kinetics of this
reaction are conveniently followed by monitoring the pseudo-
first-order rate constant of disappearance of TPZ at 470 nm as
a function of TPZ concentration. The reaction kinetics can also
be followed by monitoring the rate of formation of the reaction
product at 350 and 400 nm. The absolute second-order rate
constant for reaction of acetone ketyl radical with TPZ in
9
-1 -1
2-propanol is 9.7 × 10 M s . Similar LFP experiments with
DTBP were performed in dioxane, 1,3,5 trioxane in acetonitrile,
and diethylamine solvents. The radicals formed in these
solvents react somewhat more slowly with TPZ. It is con-
cluded that ketyl radicals react, in part, with TPZ by transferring
OH and CH hydrogen atoms from the ketyl radical to the
N-oxide oxygen atom at N4 of TPZ. These reactions form ketone
(major) and enol (minor) and a radical, TPZH, that has been
previously postulated in TPZ oxygenation chemistry.3 Radical
TPZH has a lifetime of about 1 ms in alcohol solvent and decays
by disproportionation to form TPZ and a reduced heterocyle,
which subsequently extrudes water by a polar mechanism to
form desoxytirapazamine (dTPZ). In ether-derived radicals, the
reaction proceeds by transfer of a CH hydrogen to form TPZH
and an enol ether. Hydrogen atom transfer reactions proceed at
a rate that is competitive with addition reactions. The hydrogen
atom transfer reaction likely contributes to the fast reaction of
deoxyribose radicals with TPZ in aqueous solution. The resulting
enol ethers can open and fragment by polar reactions.
The results obtained in this study are completely consistent
with the quantitative data previously reported. Greenberg, Gates,
and co-workers reported that a single-stranded DNA C1-
nucleotide radical (C1N, Scheme 3) reacts with TPZ with a rate
-5
8
-1 -1
constant of 2 × 10 M
s
in aqueous solution. This is the
same order of magnitude as the rate constants of this study.
When the radical is generated in double-stranded DNA, the
4
6
-1 -1
5
rate constant falls to 4 × 10 M s . Anderson and Denny
and co-workers also reported kinetic evidence that ribose-derived
radicals react rapidly with TPZ. Both of these two groups
favored the addition of sugar radicals to TPZ. On the basis of
4
b
isotopic labeling studies, Gates and Greenberg et al. demon-
strated that 83 ( 2% of the reaction of TPZ with a C1N radical
proceeds via addion to an N-oxide oxygen atom. We posit that
the remaining 17 ( 2% of the reaction may proceed by
â-hydrogen atom transfer. The â-hydrogen transfer mechanism
may be less important in a C1N radical than in the radical
derived from diisopropyl ether because the C1N radical has
fewer â-hydrogens available for atom transfer and the rigid
framework of the ribose ring might introduce steric and
stereoelectronic effects that retard the hydrogen atom transfer
reaction.
Although we conclude that hydrogen transfer is an important
mode of reaction, it is possible that radical addition chemistry
proceeds at a rate competitive with hydrogen transfer from a
CH group adjacent to a radical center in an ether. The hydrogen
transfer reaction leads to dTPZ as required in any mechanism,
but we posit it can also be formed by a polar mechanism which
follows a disproportionation reaction of TPZH. The proposed
reaction sequence converts the sugar radical to enol ethers,
which can open and fragment to produce DNA strand breaks
by polar mechanisms.
5. Experimental Section
5
.1. Laser Flash Photolysis Studies. The instrument used for LFP
experiments and the protocols used in these experiments are described
in detail in the literature.12 The nanosecond laser flash system used an
excimer laser (Lambda Physik LPX105EMC, 308 nm, 15 ns), or a Nd:
YAG laser (Spectra Physics LAB-150-10, ∼5 ns, 355 or 266 nm) was
used as the excitation light source. The measurement beam was supplied
by a 150 W xenon arc lamp (Applied Photophysics) used in pulsed
mode (0.5 ms in duration) with a 1 Hz repetition rate. The pulse of
light from the xenon lamp was focused onto a single ARC SP-308
monochromator/spectrograph, with a 1-015-300 grating. This model
features dual ports, one with a slit and a photomultiplier for kinetic
measurements and the other with a flat field and a Roper ICCD-Max
5
12T digital intensified charge-coupled device (ICCD) camera for
spectroscopic measurements with up to 2 ns temporal resolution. The
single monochromator/spectrograph negates the need for separate
optimization of kinetic or spectral measurement system alignment, thus
facilitating the usage of both types of measurements. The ICCD
controller is directly interfaced to the computer using the Roper
WinView software and ST-133A controller. Kinetic data acquisition
uses a Tektronix TDS 680C 5Gs/s 1 GHz oscilloscope directly
interfaced via a National Instruments PCI-GPIB to a computer
running a custom LabView control and acquisition program. Laser,
arc lamp, shutter, and other timing and control signals are routed through
a National Instruments PCI-6602 DAQ interface. The measurement
beam is supplied by a PTI 150 W xenon arc lamp with an LPS 210
power supply, LPS 221 stand-alone igniter, A-500 compact arc lamp
housing, and MCP-2010 pulser, which allows for controlled pulsing
of the arc lamp with pulses 0.5-2.0 ms in duration and up to 160 A in
2
amplitude. Samples were placed in 10 × 10 mm spectrasil quartz
cuvettes and were deoxygenated by purging with a stream of argon
for 5 min.
4
. Conclusions
(
12) (a) Gritsan, N. P.; Zhai, H. B.; Yuzawa, T.; Karweik, D.; Brooke, J.; Platz,
M. S. J. Phys. Chem. 1997, 101, 2833. (b) Martin, C. B.; Shi, X.; Tsao,
M.-L.; Karweik, D.; Brooke, J.; Hadad, C. M.; Platz, M. S. J. Phys. Chem.
B 2002, 106, 10263-10271.
Ketyl radicals were generated by laser flash photolysis (LFP)
of benzophenone or acetone in alcohols or by LFP of di-tert-
J. AM. CHEM. SOC.
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VOL. 129, NO. 15, 2007 4549